Effective Monitoring System or Anthrax, Smallpox, or Other Pathogens

ABSTRACT

A device and method for detecting anthrax or other pathogens are disclosed. Individual self-contained monitoring devices of a monitoring system can be portable or stationary (e.g. installed in air ducts or plumbing of buildings) and can be part of a network of devices. Monitoring devices may be used for the detection of a variety of airborne or surface pathogens, including but not limited to anthrax, smallpox, and  Salmonella . Bioamplification-coupled proteomics assays provide rapid and reliable detection of pathogens, with self-checking capabilities reducing or eliminating false positives and false negatives. Sample preservation capability allows pathogen samples to be preserved after detection for further testing. The device of the invention can be remotely operated by minimally trained technicians or security personnel. The pathogen monitoring device of the invention provides a more compact, accurate, rapid, and cost-effective alternative to other anthrax detectors, and an effective weapon against bioterrorism.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to provisional application No.60/337,190, filed Dec. 6, 2001 (Attorney Docket number 020054-003400US)

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

Not applicable

REFERENCE TO A “SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAMLISTING APPENDIX SUBMITTED ON A COMPACT DISK

Not applicable

BACKGROUND

The rapid detection of microorganisms, particularly highly virulentpathogens, is required for the timely treatment of serious infections.Contamination of air or water by pathogenic microorganisms can occurnaturally, can be the result of unintended human interference, or canoccur as a result of intentional use of biological warfare agentsagainst military and civilian populations. Because of the ability ofpathogens to disseminate and infect human populations rapidly, adetection system requires speed, versatility and, preferably,portability. Early detection and identification of pathogens in patientsallows a health care worker to diagnose and appropriately treat apatient. Remote sampling and detection of microorganisms can limitexposure to biological agents through the identification of contaminatedareas. These areas can then be quarantined and decontaminated byappropriately trained individuals.

However, in spite of the need for rapid detection of pathogens,detection equipment in current use has significant shortcomings.Manipulating and interpreting pathogen detection devices in the field isa hazardous duty, and can be made more difficult by cumbersomeprotective clothing worn by health care or military personnel. Thus,remote and automated sensing is required to address both safety andefficiency concerns. To be truly effective as a monitoring system, italso must be widely distributed, such that detection of bioterrorisminduced or natural outbreaks can be rapidly identified and controlled.In turn, the need for a widespread early warning network demands thatany detection device be accurate, automated and relatively inexpensive.

There are several methods commonly used to detect pathogens in collectedsamples, but not all of these methods are rapid, readily automatable orof low cost. These include (i) amplification of pathogen-specificnucleic acid sequences, including methods for amplifyingpathogen-specific nucleic acid sequences requiring numeroustime-consuming steps that are difficult to automate and often producefalse positives or false negatives; (ii) culture of pathogens onappropriate growth media, followed by isolation and eithertime-consuming biochemical or histological assays; (iii) massspectrometer-based detection of pathogen-specific components, in whicheach detection unit is expensive to produce; and (iv) serological-basedassays, which have limited sample size and can only detect pathogens inan infected individual.

There are several disadvantages to these methods. Nucleic acidamplification and many examples of biochemical assays require a methodof breaking open the cell and isolating the components. The techniquesused to break open the cell have been shown to make these assaysdifficult to reproduce and inaccurate (Dang et al., 2001, Appl EnvironMicrobiol 67(8):3665-70). Culturing pathogens on solid growth mediacan'take several days, in which time an infected patient could die or beseriously compromised. Mass spectrometry, although accurate, requireslarger sample sizes, high trained operators, and a very expensivetesting unit.

One general class of methods in comnmon use for pathogen detection,enzyme-linked immunosorbent assays (ELISAs), can be made automatable,rapid and inexpensive. With regard to the latter, low cost providesmultiple monitoring device capability. However, ELISAs are not verysensitive, and incapable of detecting small numbers (e.g., 1-10 anthraxspores) of pathogens. The method of the present invention combines thecost-efficiency and rapidity of an ELISA-based “dipstick” assay with theincreased sensitivity of bioamplification. In this manner, the method ofthe invention is able to accurately and rapidly detect very smallamounts of pathogens in airborne, fluid, or surface samples. Thesimplicity and accuracy of this protein amplification assay imparts theability to produce relatively simple and portable detection devices.

In the case of Anthrax, treatment is effective if initiated within 72hours of infection. This means that samples must be analyzed in time toidentify potentially infected individuals and begin treatment. Theinvention discloses a device that takes advantage of this latency periodto improve on the accuracy of an inexpensive test, significantlyreducing false positive and false negative results usingbioamplification. This method and device for detection of pathogens suchas Anthrax can give automated and accurate pathogen detection within12-24 hours, leaving plenty of time to begin treatment for infectedindividuals.

SUMMARY

The present invention provides a device for detection of pathogens atone or more locations. In one aspect, the invention provides stationaryor portable devices for collecting either surface or airborne samplesand determining the presence or absence of pathogens. In one embodiment,stationary detection devices can be permanently installed in air ductsfor ongoing monitoring of airborne microorganisms. In anotherembodiment, portable, devices may be used to selectively monitor areassuspected of being targets of biological warfare. In another aspect, theinvention discloses a network of collection and detection units formonitoring pathogens and potential outbreaks. Each detection unit can beeither in proximity to an assay analyzer or remotely removed from theanalyzer. A single test cartridge includes a sample chamber and a testchamber separated by a breakable seal. The sample chamber can be eithera fluid intake through which a fluidic substance, such as a gas orliquid, enters the system, or a receptacle for receiving a specimenswab, which is immersed in a fluid for suspending microorganisms.

In another aspect, the invention provides a sealable cartridge fordetecting elements of microorganisms, comprising (i) a sample chamber,wherein a test sample is suspended in fluid; (ii) a test chamber,wherein the fluid is tested for elements of a microorganism; and (iii) abreakable seal between the sample and test chambers. Some sealablecartridges further comprise a detection means. Some sealable cartridgesfurther comprise a transmission means. Some sealable cartridges furthercomprise an assay analyzer. With some such sealable cartridges, themicroorganism is Bacillus anthracis.

In another aspect, the invention further provides a pathogen monitoringsystem for detecting pathogens, comprising: an intake or receptaclethrough which a fluid or gas enters said pathogen monitoring system oris added to said pathogen monitoring system; a means for drawing orpushing said fluid or gas into said pathogen monitoring system andthrough a conduit connectively communicating with said intake; dfiltering means within said conduit through which said fluid or gaspasses and retains microorganisms present in said fluid or gas; a mediumreservoir for a growth medium on a side of said filtering means oppositefrom said intake or receptacle, wherein said growth medium passesthrough said filtering means and removes said microorganisms from saidfiltering means. Some pathogen monitoring systems further comprise adetection means. Some pathogen monitoring systems further comprise atransmission means. Some pathogen monitoring systems further comprise anassay analyzer. With some pathogen monitoring systems, the microorganismbeing monitored is Bacillus anthracis. In one embodiment, the pathogenmonitoring system may be used for the detection of microorganisms beingused or suspected of being used for bioterrorism.

In another aspect, the invention further provides a pathogen monitoringsystem for detecting microorganisms, comprising: an intake or receptaclethrough which a fluid or gas enters said pathogen monitoring system oris added to said pathogen monitoring system; a means for drawing orpushing said fluid or gas into said microbial monitoring system andthrough a conduit connectively communicating with said intake; afiltering means within said conduit through which said fluid or gaspasses and retains microorganisms present in said fluid or gas; anexhaust chamber, wherein said filtering means is interposed between saidintake and said exhaust chamber; a medium reservoir on a side of saidfiltering means opposite said retained microorganisms, comprising agrowth medium and a reservoir breakable seal; a plunger means, whereinpressure placed on said plunger means: (i) engages a hermetic sealbetween said medium reservoir said filtering means; (ii) breaks saidreservoir breakable seal and forces growth medium through said filteringmeans in a direction opposite of flow of said fluid or gas; and (iii)backwashes said microorganisms from said filtering means and suspendssaid microorganisms in said growth medium; an incubation chamber forincubating and growing said microorganisms; a programmabletemperature-regulating means for controlling the temperature of saidincubation chamber; an incubation chamber breakable seal through whichsaid growth medium passes into a test chamber for detecting saidmicroorganisms; a timer for controlling intervals between assaymeasurements; a detection means within said test chamber; wherein saiddetection means determines the presence or absence of saidmicroorganisms without killing said microorganisms or inhibiting saidgrowing; and a transmission means; wherein said presence or absence ofsaid microorganisms is reported via said transmission means to an assayanalyzer. The simplicity of the device and the use of a simple positiveor negative (i.e. presence or absence) result allows the user toeffectively operate the device with little training, thereby reducingoperating costs.

With some pathogen monitoring systems, the transmission means is a wireconnection, a radio link, a microwave transmission, an infraredtransmission, a cellular phone, or a computer network. With somepathogen monitoring systems, the pathogen monitoring system comprises anetwork of separate monitoring devices. With some such pathogenmonitoring systems, the assay analyzer is remote from said pathogenmonitoring system and said reporting relays location of a pathogenmonitoring device to said assay analyzer. With some such pathogenmonitoring systems, the location is determined by a global positioningsystem. With some pathogen monitoring systems, the detection means isselected from the group consisting of an enzyme immunoassay, an opticalimmunoassay, mass spectroscopy, and gene sequence analysis. With somesuch pathogen monitoring systems, the enzyme immunoassay comprises animmunoassay strip that wicks the growth medium past a detection area anda control area on said detection strip.

With some microbial monitoring systems, the microorganisms beingmonitored are selected from the group comprising of Bacillus anthracis,Yersinia pestis, Neiserria menigitidis, Coxiella burnettii, Coccidiodesimmitis, Francisella tularensis, Cryptcoccosis neofomans, Escherichiacoli, Haemophilus influenzae, Brucella species, Salmonella species,Shigella species, Chlamydia psittaci, Vibrio cholerae, Staphylococcusenterotoxin B, Histoplasma capsulatum, Rickettsia species,Corynebacterium diphtheriae, Burkholderia pseudomallei, Burkholderiamallei, Mycobacterium tuberculosis, Blastomyces dermatitidis, andNocarida species.

With some pathogen monitoring systems the pathogen monitoring systemcomprises an optical sensor for determining assay results. With somepathogen monitoring systems, the pathogen monitoring system comprises avariable timer that determines the frequency of detection assays. Withsome pathogen monitoring systems, the pathogen monitoring systemcomprises a detection unit comprising a disposable cartridge. With somesuch pathogen monitoring systems, the disposable cartridge is moved inautomated fashion into place for incubation and assay determination, thedetecting is conducted automatically, and the disposable cartridge isautomatically removed to a waste chamber after said detecting. With somepathogen monitoring systems, the plunger means comprises a bladed orpointed edge for releasing said growth medium. With some pathogenmonitoring systems, the programmable temperature-regulating meanselevates the temperature of the microbial monitoring system to shockbacterial endospores and initiate endospore germination.

In another aspect, the invention further provides a pathogen monitoringsystem for detecting microorganisms, comprising: a network of pathogenmonitoring devices reporting to a remote assay analyzer, wherein each ofsaid pathogen monitoring devices comprises at least one disposablecartridges comprising: an intake or receptacle through which a fluid orgas enters said pathogen monitoring system or a swab is deposited withinsaid pathogen monitoring system in a fluid; a means for drawing orpushing said fluid or gas into said pathogen monitoring system andthrough a conduit connectively communicating with said intake orreceptacle; a semipermeable membrane within said conduit through whichsaid fluid or gas passes and which retains microorganisms present insaid fluid or gas; an exhaust chamber, wherein said semipermeablemembrane is interposed between said intake and said exhaust chamber; amedium reservoir containing growth medium on the side of saidsemipermeable membrane opposite said retained microorganisms; a plungermeans comprising a bladed or pointed edge for releasing said growthmedium; wherein depressing said plunger means: (i) engages a hermeticseal between said medium reservoir said semipermeable membrane; (ii)forces growth medium through said semipermeable membrane in a directionopposite of flow of said fluid or gas; and (iii) backwashes saidmicroorganisms from said semipermeable membrane and suspends saidmicroorganisms in said growth medium; a breakable seal through whichsaid growth medium passes into a test chamber for incubating and growingsaid microorganisms; a timer for determining intervals betweenimmunoassay measurements; a temperature-regulating means that maintainssaid test chamber at a temperature that promotes growth of at least onetarget microorganism; an immunoassay test strip within said testchamber; wherein said immunoassay strip wicks said growth medium past adetection area on said detection strip, said detection area comprises anindicator assay region and a control assay region; and said immunoassaytest strip determines the presence or absence of said at least onetarget microorganism, wherein said presence or absence of said at leastone target microorganism is reported with location of said microbialmonitoring device to said remote assay analyzer.

In another aspect, the invention further provides a method for detectingpathogens in an air or fluid specimen, comprising: passing an air or gasspecimen or a specimen derived from a swab through a pathogen monitoringsystem, wherein the pathogen monitoring system comprises an intake orreceptacle through which a fluid or gas enters said pathogen monitoringsystem or is added to said pathogen monitoring system; a means fordrawing or pushing said fluid or gas into said pathogen monitoringsystem and through a conduit connectively communicating with saidintake; a filtering means within said conduit through which said fluidor gas passes and retains microorganisms present in said fluid or gas; amedium reservoir for a growth medium on a side of said filtering meansopposite from said intake or receptacle, wherein said growth mediumpasses through said filtering means and removes said microorganisms fromsaid filtering means; retaining microorganisms within said air or fluidspecimen on a filtering means within said pathogen monitoring system;incubating said microorganisms with a growth medium in said pathogenmonitoring system at temperature that promotes growth of at least onetarget microorganism; and determining presence or absence of said atleast one target microorganism with a detection means within saidpathogen monitoring system; where determination of the absence of saidat least one target microorganism initiates a subsequent round ofincubation and determining.

In another aspect, the invention further provides a kit for detectingpathogens, comprising: at least one disposable cartridge for use in apathogen monitoring system, wherein the pathogen monitoring systemcomprises an intake or receptacle through which a fluid or gas enterssaid pathogen monitoring system or is added to said pathogen monitoringsystem; a means for drawing or pushing said fluid or gas into saidpathogen monitoring system and through a conduit connectivelycommunicating with said intake; a filtering means within said conduitthrough which said fluid or gas passes and retains microorganismspresent in said fluid or gas; a medium reservoir for a growth medium ona side of said filtering means opposite from said intake or receptacle,wherein said growth medium passes through said filtering means andremoves said microorganisms from said filtering means; and a sterilecontainer for retaining said at least one disposable cartridge.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows an exploded view of one embodiment of a test cartridge.

FIG. 2 shows an assembled view of the test cartridge.

FIG. 3 shows the path of airflow through the test cartridge.

FIG. 4 shows a cross section of the test cartridge in sampling position.

FIG. 5 shows a cross section of the test cartridge following airsampling and sealing.

FIG. 6 shows a cross section of the test cartridge after plunger as beendepressed forcing the growth medium through the filter into the growthchamber.

FIG. 7 shows the test strip and its holder.

FIG. 8 shows one embodiment of the automated system that moves, heats,times, senses changes in, records data from and stores the testcartridges. Numbered parts as follows: 1—Delivery cassette, 2—Incomingcartridge, 3—Cartridge feed mechanism, 4 —Feedmotor, 5—Sampling station,6—Air intake, 6′—Air exhaust, 7—Plunging mechanism, 8—Incubationstation, 9—Heat control card, 10—Result sensor card, 11—Outgoingcartridge position holder, 12—Cartridge ejector solenoid, 13—Biologicalwaste cassette, 14—Microcontroller card.

FIG. 9 shows an exploded view of a different embodiment of the testcartridge for use with manually loaded swabs.

FIG. 10 shows a portable device for testing six manually loadedcartridges.

FIG. 11 shows another embodiment of the test cartridge with multipledetection strips.

FIG. 12 shows another embodiment of the automated system that moves,heats, times, senses changes in, records data from multiple testcartridges running concurrently and stores the test cartridges. Numberedparts as follows: 1—Delivery cassette, 2—Incoming cartridge, 3—Cartridgefeed mechanism, 5—Sampling station, 6—Air intake and exhaust,16—Carousel, 17—Cartridge with one test left, 18—Plunge and readstation, 19—Plunge and read station for 3rd test.

DETAILED DESCRIPTION OF THE INVENTION I. General

The terms “fluid” or “fluidic substance” as used herein refers to anyfluid, including air, a gas, or a liquid, including water and an aqueoussolution.

The term “target microorganism” as used herein refers to a particularmicroorganism that one wishes to detect in a fluid sample. Targetmicroorganisms can include pathogens, including bacterial, protozoal,viral, and fungal pathogens. Target microorganisms can be naturally,accidentally, or intentionally introduced into a medium, such as air ora liquid plumbing system or reservoir, from which the fluid sample isderived. Target organisms can include those found in clinical settingsthat can cause nosocomial infections, such as Enterobacteriaceae,Pseudomonas species, Streptococcus species, and Staphylococcus species,and can include biowarfare agents and potential biowarfare agents.Examples of biowarfare agents or potential biowarfare agents includeBacillus anthracis, Burkholderia mallei, Burkholderia pseudomallei,Brucella species, Chlamydia psittaci, Corynebacterium diphtheriae,Coxiella burnettii, Cryptococcosis neofomans, Escherichia coli,Francisella tularensis, Haemophilus influenzae, Mycobacteriumtuberculosis, Neiserria menigitidis, Rickettsia species, Salmonellaspecies, Shigella species, Staphylococcus species, Streptococcusspecies, Vibrio cholerae, and Yersinia pestis. Target microorganisms canalso include fungal pathogens that can be naturally present in anenvironment, or intentionally introduced as biowarfare agents. Examplesof the latter include Blastomyces dermatitidis, Coccidiodes immitis,Histoplasma capsulatum, and Nocarida species. “Elements” of amicroorganism include, but are not limited to, proteins, nucleic acids,secreted toxins, and alike, of a microorganism. “Elements” of amicroorganism can also include any identifiable feature of amicroorganism.

The term “monitoring device” refers to a single unit that can be fixedor portable, is programmable with respect to fluid sampling and assayparameters, and which contains the components necessary to determine thepresence of microorganisms. The components include a power supply (ifthe unit is portable), a sampling station, one or more test cartridges,control and test result sensing elements.

The terms “microbial monitoring system” and “pathogen monitoring system”refers to one or more monitoring devices. If more than one monitoringdevice is used, they can be linked in a network that provides detectiondata to a single assay analyzer.

The term “detection unit” refers to a device for manipulation of testcartridges and sensing results of test cartridge assays. Detection unitcan be used interchangeably with “monitoring device”.

The terms “growth medium” and “growth media” refer to a mixture ofnutrients that will support the growth of the target microorganism. Thiscan include vitamins, minerals, salts, proteins, nucleic acids, yeastextracts, bacterial extracts and appropriate cells to allow growth ofparasitic or pathogenic microorganisms.

The term “test cartridge” refers to a sealable chamber that includes ameans for suspending a sample in liquid (sample chamber), a chamber witha detection strip or similar means to assay for the presence of amicrobial element (test chamber), and a breakable seal between saidelements.

The term “monitoring device” can refer to a detection unit or pluralityof detection units.

The term “assay analyzer” refers to the test result-sensing unit of themonitoring device or a plurality of sensing units.

The term “pathogen” refers to any biological organism that can bepropagated in humans or animals.

II. Overview

The invention is a monitoring system for detecting the presence ofpathogens, including pathogenic microorganisms that can be present in anairborne, fluidic, or surface sample. The device comprises a sealablecartridge for sample collection and testing, and test result sensingunit. The device also comprises at least one element for reporting thetest results. Multiple device units can constitute a network of devicesreporting to a centralized location.

Each monitoring device further comprises at least one detection unit.The detection unit can comprise a test cartridge that can be disposable.Each monitoring device can also comprise multiple detection units. Oneor more detection units can be placed either in proximity to an assayanalyzer, which is able to evaluate data from one or more detectionunits, or can be remotely removed from the analyzer. The evaluation ofthe data transmitted to the assay analyzer includes an assessment ofwhether one or more target microorganisms are present in a sample of afluid.

The invention finds use in hospitals, laboratories or other buildingswhere pathogenic microorganisms can be present in, for example, air,plumbing, water specimens or water sources to which pathogenicmicroorganisms can be accidentally or intentionally introduced. The lowcost, short time to assay results, the potential portability and thewide variety of pathogens that can be detected with the presentinvention allows a large number of monitoring devices to be used over awide area for comprehensive detection capability.

In one embodiment, the invention may be used in areas suspected of beingtargets for bioterrorism. Stationary monitoring devices of the inventionmay be installed in air ducts or plumbing of buildings that may betargets of biological warfare (e.g. post offices, government offices,heavily populated buildings) to constantly monitor levels of pathogensthat may be used as biological weapons (e.g. anthrax). Portablemonitoring devices may be used at multiple locations to monitor levelsof pathogens in a wide region.

The invention may be operated remotely and results may be transmitted toa remote location, such that operators of the device will not riskexposure to the pathogen in the course of monitoring. This reduces costsignificantly by eliminating the need for costly biological hazard teamsand equipment currently used to enter potentially contaminated areas anddetect the presence of pathogens at those locations.

III. Exemplary Monitoring Devices

One advantage of the presently disclosed monitoring system is theprogrammable nature of the system. The amount of fluid that enters themonitoring system, the temperature of incubation, the duration ofincubation, the number of detection assays performed, and, optionally,the duration and temperature for “heat-shocking” bacterial endosporesare all controllable parameters that can be programmed in advance oraltered once a fluid has entered the monitoring device. The monitoringsystem is also versatile, in that it can be used to detectmicroorganisms in a gas, such as air, in a fluid, such as a waterspecimen, or obtained from a sample swab that has been wiped across asurface. A single monitoring device can be used to perform a pluralityof assays, with either one specimen tested multiple times, or a numberof fluid specimens tested. Another advantage of the present monitoringsystem is that the disclosed devices can be remote from a single assayanalyzer that is used to report assay results. Another advantageincludes the ability to link separate and remote monitoring devices in anetwork, thus providing the ability to detect target microorganismsacross a selected region.

A single detection unit includes a sample chamber and a test chamberseparated by a breakable seal (FIGS. 4 and 9). The sample chamberincludes either a fluid intake through which a fluid enters thedetection unit, or a receptacle for receiving a sample swab used forsurface testing. The ability of the unit to be adapted to airborne,fluidic, or surface pathogens provides a distinct advantage over othermicroorganism detectors. In the latter case, a suspending fluid is addedto the receptacle to suspend microorganisms, including targetmicroorganisms, which can be present on the swab (FIG. 9). Movement ofthe fluid into the sample chamber of the test cartridge can be generatedby pressure, vacuum, electrokinetics, capillary action, gravity, manualactions or a combination thereof. Gas specimens are preferably drawninto the fluid intake by a vacuum pump located at or near and operablyconnected to an outlet of the test cartridge (FIG. 3). In the embodimentfor gas or, fluid samples, the fluid passes from the intake e through aconduit that leads to a filtration means, which can include asemipermeable membrane (FIGS. 3 and 4). A semipermeable membrane used inthe present invention is of sufficient pore size and density such thatthe membrane retains microorganisms present in the fluid but also allowsthe fluid to pass through the membrane at an acceptable flow rate. Themembrane can be composed of a number of materials routinely used in theart, including nitrocellulose, polycarbonate, nylon, cellulose acetate,coated cellulose acetate, polyethersulfone, polyester, glass fiber,polyethersulfone, polypropylene, polytetrafluoroethylene (PTFE;Teflon®), polyvinylidenedifloride (PVDF), and copolymers. Typically,membrane mean pore size will be on the order of 0.1 to 5 microns,preferably 1 to 5 micron pore size for retaining protozoa and fungi, andmore preferably 0.2 to 0.5 microns for retaining bacteria. Acceptableflow rates are typically greater than 1 liter/minute for gaseous samplesand 10 milliliters/minute for liquid samples.

The fluid passes through the membrane and exits through an exhaustchamber around a medium reservoir (FIG. 3). The medium reservoircontains a growth medium that is able to support the growth of a targetmicroorganism. Typically, growth media used in this invention will becomplex media that contain nutrients, vitamins, minerals and variousother substances that allow for the rapid growth of one or more targetmicroorganisms. The medium reservoir consists of a closed container withat least one seal that can be readily pierced or broken to release thegrowth medium contained within the medium reservoir. The growth mediumwithin the reservoir will typically be sterile. Examples of growth mediathat can be used in this invention include general purpose ornon-selective media such as nutrient broth, Sabouraud culture media,trypticase soy broth, and brain-heart infusion media. Selective mediacan be used in the present invention to suppress or inhibit the growthof non-target microorganisms while permitting the growth of targetmicroorganisms. Examples of selective media include those containingantibiotics, dyes such as crystal violet, or azide that inhibit or killnon-target microorganisms. Selective media can also comprise a selectivepH or electrolyte concentration. Enrichment media can be used in thepresent invention to suppress the growth of competitive normalmicroflora while enhancing the growth of target microorganisms. Examplesof enrichment media include media enhanced by addition of bicarbonate tothe growth medium for enriching for Bacillus anthracis (e.g., 0.8%sodium bicarbonate in the presence of 5% CO₂), GN broth, Hajna (e.g.,for enrichment of Shigella or Salmonella), selenite crystal broth (forenriching for Salmonella, and tetrathionate broth (for enriching forSalmonella). Other specialized isolation media can be used in thepresent invention, such as, for example, Lowenstein Medium for theselection an enrichment of mycobacterial species.

Test cartridges for liquid or gas sample collection further comprise aplunging means for piercing or breaking a seal of the medium reservoir(FIG. 6). The component has a knife or pointed edge that, when itcontacts the medium reservoir seal with sufficient force, pierces orbreaks the seal and releases the growth medium. Thus, when the plungermeans is depressed, the growth medium is released from the mediumreservoir. Depressing the plunger means also and simultaneously engagesa seal between the medium reservoir and the semipermeable membrane. Insome embodiments, this seal can be hermetic. By the action of theplunger means, the growth medium present in the reservoir is forcedthrough the membrane in a direction opposite of the flow of the fluidsample. This has the effect of backwashing any microorganisms that canbe retained on side of the semipermeable membrane opposite the mediumreservoir and suspending the microorganisms in the growth medium.

The test cartridge also contains a sample chamber that receives thegrowth medium from the filter. In a preferred embodiment, microorganismscaptured by the membrane are able to grow in the medium, metabolize andproduce specific analytes. The movement of the growth medium into thechamber can be generated by pressure, vacuum, electrokinetics, capillaryaction, gravity, or a combination thereof. A breakable seal isinterposed between the sample chamber and a test chamber, the lattercontaining a means for the detection of the target organism. Movement offluid from the sample chamber to the test chamber following breakage ofthe seal between the chambers can be generated by gravity, vacuum,capillary action or other means.

In one embodiment, a sufficient number of target microorganisms areoriginally present in a fluid specimen that a period of growth andassociated biological amplification is not necessary to detect thetarget microorganisms. In this embodiment, the microorganisms aredetected during a first detection assay, as described generally and inspecific embodiments below.

The device also comprises a programmable temperature-regulating meansthat maintains the sample chamber at a temperature that promotes growthof at least one of the target microorganisms that can be present in thegrowth medium. The growth medium is incubated for a period of time andthen assayed for the presence of at least one of the targetmicroorganisms, as described below. Programmable temperature-regulatingmeans used in the monitoring device are well known in the art, and in apreferred embodiment include electric resistive heating elements. Thetemperature of the chamber is generally adjusted to an optimum or nearoptimum temperature for growth of one or more of the targetmicroorganisms. Incubation temperatures vary depending on therequirements of the target microorganisms, and are typically within therange of 12° C. to 45° C. The temperature for optimizing growth ofEnterobacteriaceae, including Shigella, Escherichia, and Salmonellaspecies, is preferably about 37° C. The temperature for optimizing thegrowth of Bacillus anthracis is preferably about 35° C.

At least one programmable timer is used in the monitoring device toregulate and control the duration of the intervals between assaymeasurements. At least one timer can be mechanical, and preferablyelectronic or computer-based. At least one timer can also be variablyprogrammable so that time intervals of various durations can beprogrammed, such as, for example, one length of time for heat-shockingendospores and another time length of time for incubating microorganismsat an optimum or near-optimum temperature for growth. The time andduration of elevated temperature treatment can vary according to thetarget microorganism being detected. For example, endospores of B.anthracis can be incubated at 65° C. or 75° C. for 20 minutes to inducegermination of endospores, although the temperature and duration of theshock period can vary with specific requirements of suspected targetstrains.

The timer can be used to control intervals between detection assays inthe monitoring device; a negative assay result is followed by subsequentround of incubation of the growth medium in the detection unit andanother detection assay after a suitable period of time has elapsed. Theperiod of incubation will vary depending on the type of microorganismbeing detected. The incubation periods for assays for the detection offast growing species such as Bacillus anthracis or Salmonella specieswould typically be of short duration, such as, for example, about 10 to120 minutes. The incubation periods for the detection of slow growingspecies such as Mycobacterium species are typically of longer duration,and can be from about 1 to 24 hours. The monitoring device can also bemanually directed, either at a remote control unit or at the monitoringdevice, to repeat an incubation period and assay measurement. Theincubation allows the unit to determine the presence or absence of apathogen in the sample in a relatively small amount of time, typically 6to 24 hours. If no incubation period is required (e.g., if theconcentration of pathogen exceeds levels for which incubation isrequired), the response time of the detection unit may be reduced to aslittle as 10 minutes.

In embodiments where the test sample is being assayed for presence of avirus, the growth media may include an appropriate viral host organismfor bioamplification.

In the case of Bacillus anthracis, infected individuals may becomeseverely compromised in as little as 3 days. The 24 hour maximumresponse time of the detection device allows health care workers toeffectively treat patients well before symptoms may become severe orlife-threatening.

As microorganisms grow and metabolize, they release or secretesubstances that can serve as potential analytes for a detection assay.Examples of such analytes that can be used to identify microorganisms ina protein-based assay include proteins and peptides such as Bacillusanthracis toxin, Yersinia outer membrane proteins, Staphylococcusenterotoxin B, Blastomyces dermatitis, WI-1 adhesive, Histoplasmacapsulatum H antigen, and cholera toxin. A test chamber forproteomic-based assays comprises a means for the detection of at leastone target microorganism analyte. For example, a single-step enzymeimmunoassay can be used for detection. In this embodiment, the testingmeans includes a strip composed of a adsorptive material that is capableof wicking by capillary action a liquid to a capture and test orindicator region on the detection strip (FIG. 1). The adsorptivematerial permits the adsorptive transport of the growth medium betweenthe test chamber and the control test regions. When the growth mediumfrom the test chamber moves to the either region, analytes released orsecreted by target microorganisms bind to the capture region. Thepresence of microorganisms in the growth medium in minimally detectablenumbers is determined with a detection means in the test chamber.Because a small fractional of the growth medium is removed from the testchamber, the detection means does not significantly affect the volumegrowth medium, which is available for subsequent incubation anddetection assays.

In one embodiment, the sample in growth medium may be preserved forfuture detection assays. Preservation may be achieved via freezing ofthe detection unit, isolation of the sample and growth medium forinoculation on petri dishes, or other methods known in the art.

In one embodiment, the detection means of the test chamber comprisesmonoclonal anti-analyte antibodies bound to a colored pigment. After theseal between the sample chamber and the test chamber is broken, growthmedium enters the test chamber and specific microbial analytes that havebeen generated and/or released into the medium by target microorganismsbind the anti-analyte antibodies on the detection strip. Theanalyte-pigmented antibody complex contacts the sorptive material of thetest strip and, by capillary action, moves to the test and controlregions of the strip. In the control region, polyclonal anti-mouseantibody is immobilized on the strip. By virtue of the presence of mouseantibody, mouse antibody:analyte complex binds to the control region andacts as a positive control. The test region of the test strip containsimmobilized polyclonal anti-analyte antibodies bound to the strip. Byvirtue of a specific binding interaction of the bound antibody andanalyte, mouse antibody:analyte complex, if present, binds to theimmobilized polyclonal anti-analyte antibodies of the test region. Anoptical sensor (FIG. 8, feature 10), generally located outside of thetest chamber, the latter having at least one exterior transparentcomponent, can be used to determine the presence of pigment at the testand control regions of the detection strip. The presence or absence ofpigment at the test region is reported to the assay analyzer as apositive or negative result, respectively.

In various other embodiments of the invention, the detection means ofthe test chamber comprises a method of detecting the presence or absenceof a pathogen using fluorescence, calorimetric analysis, radioactivetagging, magnetic tagging, or other detection means known in the art.

The monitoring device can be programmed with a control unit, preferablya computer control unit. The control unit is used to program variousassay parameters, including sampling parameters such as quantity, flowrate and duration of sampling, plunger activation, incubationtemperatures, incubation periods, incubation chamber seal puncture andnumber of assay cycles. The control unit can also be used to direct themovement of one or more detection chambers within the monitoring device.The control unit can be built into the monitoring device, can be locatedin the proximity of the monitoring device, or can be remote.Instructions for the monitoring device can also be transmitted over acomputer network. The use of a computer control unit allows a humanoperator to program initially or change assay parameters as desired orrequired.

A preferred embodiment of the monitoring device is shown in FIGS. 1through 8. The following numbers refer to features on FIG. 8 unlessotherwise indicated. A delivery cassette (1) contains test cartridges(2), which are moved to sampling station (5) via a cartridge feedmechanism (3) operably linked to a feed motor (4). An air sample inpulled through the monitoring device using an air intake (6) thatdirects the airflow through the test cartridge (FIG. 4) and exits themonitoring device through the exhaust (6). A plunging mechanism (7) thenseals the test cartridge (FIG. 5). The plunger then depresses the growthmedia cartridge which releases the media through the collection filter(FIG. 6) and into bottom of the sample chamber. Test cartridges aremoved to an incubation station (9) which holds each test cartridge at atemperature appropriate for the growth of the target organisms. Thetemperature within the incubation station is controlled with a heatcontrol card (9). After a programmed incubation period, a breakable seal(FIG. 4) is pierced and growth medium enters a test chamber (FIG. 4).The medium moves up a detection strip (FIG. 1) within the test chamberand past the indicator and control regions of the detection strip (FIG.4), at which antibody:analyte complexes will bind. Multiple detectionstrips can be placed in the same cartridge. After the detection stripassay is performed within the test chamber, results are detected by theresult sensor (10). The test cartridge is subsequently moved to theoutgoing cartridge position holder (11) then by the cartridge ejectorsolenoid (12) into the biowaste cassette (13). Means for transmittingprogrammed instructions from the control unit to a remote monitoringdevice include wire connection, a radio link, phone lines, microwavetransmission, infrared transmission, a cellular phone, or a computernetwork.

In another preferred embodiment of a test cartridge operating device,the device can test a plurality of test cartridges concurrently (FIG.12). FIG. 12 demonstrates a device capable of heating, incubating, andsensing test results from 8 test cartridges. Each of the test cartridgesshown in this example has 3 testing chambers (FIG. 11), with breakableseals at the base of the testing chambers and a non-opaque window forviewing test results on the side or the top of the chamber. Thesechambers can be used to test samples at different time points ordetection strip tests that are incompatible for concurrent testing.

Assay results, i.e., the presence or absence of target microorganisms,can be displayed at the device but preferably are reported to a remoteassay analyzer. Typically, the assay analyzer is a computer ormicrocomputer and preferably is the same computer or microcomputerfunctioning as the control unit. The assay analyzer is used for bothsignal processing and data storage. Means for transmitting assay resultsfrom the monitoring device to an assay analyzer include wire connection,a radio link, phone lines, microwave transmission, infraredtransmission, a cellular phone, or a computer network. The assay resultscan also be transmitted over a computer network.

If more than one monitoring device is used, any one device can identifyits location to the assay analyzer with positional information derivedfrom manual data entry or, preferably, from a global positioning meansthat automatically communicates the latitude and longitude coordinatesof the monitoring device. The positional information can be communicatedto the assay analyzer via wire, microwave, radio, or cellular telephonetransmission, and can be transmitted along with assay results. The assayanalyzer is capable of storing and reporting location information forall of the monitoring devices used within a defined geographical regionbeing tested for the presence of microorganisms. Limitations as to thenumber of monitoring devices communicating with a single assay analyzer,and the size of the geographical region being tested, are determined bycomputer's power and data storage capacity, and transmission means,respectively. Given the capacity of today's computers to process andstore large quantities of data and transmit data at high speeds, it isanticipated that a large number of individual monitoring devices can belinked to a single assay analyzer. In regions where data transmissionfactors limit the number or distribution of monitoring devices, multiplenetworks of device, each network having a single assay analyzer, can beemployed. In the latter case, and where data transmission is via radioor microwave, monitoring devices and control units can also transmit aregistration signal to avoid “cross-talk” between different networks ofdevices.

For any one monitoring device, assay results are reported to the assayanalyzer as a positive result, i.e., a target microorganism is presentin at least one test chamber, or as a negative result, i.e., no targetmicroorganism has been detected in a given test chamber. The assayanalyzer can also have the ability to determine and update the status ofany and all monitoring devices with which it is communicating. In thisembodiment, the monitoring device signals the assay analyzer, eitherafter a query or at regular intervals, that the monitoring device isperforming within specified parameters. The monitoring device can beequipped with sensors that report internal temperature settings, plungeractivation and fluid and detection unit movement.

Prior to the termination of assay measurements, the reporting of anegative result to the assay analyzer can prompt a subsequent round ofincubation of the growth medium and a subsequent detection assay. Anyone monitoring device can contain a plurality of detection units forperforming additional detection assays after negative results with onedetection unit are reported to the assay analyzer. In one embodiment,fluid specimens are similarly passed through multiple test units ratherthan a single test unit. Following a prescribed incubation period andnegative assay result within a first test unit, the breakable sealbetween the incubation chamber and the test chamber of a second testunit can be pierced and a second detection assay performed. This cyclecan be repeated until such time as a positive result is reported, or alldetection units through which the fluid specimens passed have been used,or a period of time has elapsed without positive detection that suggeststhat no target organism existed in any of the fluid specimens. In asecond embodiment, a single monitoring device can be used in a detectionassay for one fluid or swab specimen, after which the detection unitsused in that determination are disposed, and new detection units aremoved into place to detect microorganisms in a distinct specimen. Inthis manner, the detection device reduces or eliminates the occurrenceof false positives or false negatives via the ability to repeatdetection tests at a given location.

Because each monitoring device contains a plurality of detection units,each monitoring device can also be used to assay distinct fluidspecimens. After any detection unit within a monitoring device has beeninoculated and assayed, separate detection units can be inoculated withfresh fluid specimens derived from air, liquid or swab samples.

In one embodiment, the plurality of detection units may each be directedtowards the detection of a specific pathogen different from thepathogens detected by the other units in the plurality. In this way, theplurality may be used to detect the presence or absence of severaldifferent microorganisms at one location.

In another embodiment, the plurality of detection units may all bedirected towards the same pathogen (e.g. anthrax). In this way, theplurality may be used to detect the presence or absence of one pathogenat many locations, or may use a self-checking mechanism of repeatedlytesting for one pathogen at the same location.

Due to the small size and low energy requirements of an individualmonitoring device, the device can be made portable. For portable units,location information is preferably determined by global positioningmeans and transmitted to the assay analyzer via microwave, radio, orcellular telephone transmission.

The invention is also directed to methods for detecting pathogens in anair or fluid specimen. These methods include passing a fluidic sample,such as a gas or liquid specimen, through the pathogen monitoring systemdescribed herein. Microorganisms that are present in the sample areretained on a semipermeable membrane. The microorganisms are incubatedin a growth medium suitable for at least one target microorganism thatcan be present in the specimen at a temperature appropriate for growingat least one of the target microorganisms. The presence or absence oftarget microorganisms is determined with a detection means within thepathogen monitoring system. A negative result, that is, failure todetect a target microorganism in any one detection assay, prompts asubsequent round of medium incubation and detection assay, until suchtime that one or more target microorganisms are detected, or after asufficient period has elapsed in which one or more target microorganismswould have grown to detectable numbers if present in the specimen.

The invention is also directed to a kit for detecting microorganisms.The kit comprises at least one disposable cartridge for use in themicrobial monitoring system disclosed herein, and at least one sterilecontainer for retaining the disposable cartridges. Kits typicallyinclude a label indicating the intended use of the contents of the kit.The term label includes any writing, or recorded material supplied on orwith the kit, or which otherwise accompanies the kit.

The invention is also directed at early detection of pathogeniccontamination. Automated monitoring devices can be installed in a widevariety of locations such as post offices, commercial centers,government buildings, airplanes, transportation centers (i.e., airports,bus terminals, train stations, and shipping docks and alike), and otherlocations suspected of being likely targets of bioterrorism. Thesedevices can be programmed to run a test using an appropriate testcartridge at predetermined intervals. Results from these tests, alongwith device location information, can be sensed and reported to theappropriate agency. Appropriate agencies can include Centers For DiseaseControl and Prevention, Office of Homeland Security, medicalprofessionals, and hazardous response teams.

EXAMPLES Example 1 Analysis of Manually Collected Samples for Bacillusanthracis Spores

This example illustrates the use of a device such as that represented inFIGS. 9 and 10. First, surfaces to be examined for the presence ofBacillus anthracis should be wiped with an appropriate collector such asa cotton swab or a small filter. The collector should then be placed inthe side of the cartridge that will receive the growth media insert asillustrated in FIG. 9, such that the collector rests below the ‘Fill toHere’ line indicated on the drawing. The growth media and detectionstrip inserts should be placed into the cartridge, and the cartridgeloaded into a slot of the device shown in FIG. 10.

In this case, we are testing for Bacillus anthracis spores. The mediashould be appropriate for the germination of spores followed by growth,such as CA broth (Thorne and Belton, 1957, J. Gen. Microbiol.17:505-516) supplemented with 20 ug/ml tryptophan and buffered with 100mM HEPES pH 8.0 (Dai and Koehler, 1997. Infection and Immunity65:2576-82). The program for the machine should be set to heat shock thecollected spores for 20 minutes at 65° C., followed by a reduction intemperature to 37° C. to allow for growth of bacteria. Incubation at 37°C. should continue for two hours or for a length of time such that thenumber bacteria produce enough toxin to be above the detection limit ofthe detection strip.

At the end of the bacterial growth stage, the device of FIG. 10 willbreak the breakable seal between the chambers of the cartridge (FIG. 9).The bacterial suspension will then flow into the chamber containing thedetection strip specific for an element of Bacillus anthracis. Once incontact with the detection strip, the suspension will be wicked up thelength of the strip past the indicator and control lines. This processshould take approximately 3-5 minutes. The results will be analyzed bythe result sensor, which will compare the indicator line to the positivecontrol. If the indicator line and the control line are positive, thetest will indicate the presence of Bacillus anthracis spores in thecollected sample. If the indicator line is negative and the positivecontrol line is positive, then no Bacillus anthracis spores werecollected in the sample. If the positive control line is negative forany reason, the test is designated as a failure and should be repeated.

Results from this test will be displayed as appropriate for thesituation. This could be through digital or printed results displayed atthe device, or through an integrated communication system such as a cellphone to a remote computer. In another aspect, a coupled GPS receivercould identify the location of the unit upon initiation of the sampletesting, and communicate this information as well.

Example 2 Analysis of Air Samples for Bacillus anthracis Spores

This example illustrates the use of a device such as that represented inFIG. 8 using cartridges such as that shown in FIGS. 1 through 7. Thedevice can be placed in any upright position such that the inlet (6) andoutlet (8) of FIG. 8 are not obstructed and have air access. Cartridgesspecific for detection of elements of Bacillus anthracis should beloaded into the delivery cassette (FIG. 8-1). The machine can beprogrammed to activate a detection run either remotely or on a schedule.

Upon activation of a run, a test cartridge is moved into the samplingstation (FIG. 8-5) by the cartridge feed mechanism (FIG. 8-3). The fan(FIG. 8-6′) is activated and air is pulled through the cartridge (seeFIG. 3) from the air inlet (FIG. 8-6) and exhausted through the exhaustport (FIG. 8-6′). Approximately 15 liters of air should be pulledthrough the cartridge (equivalent to 2 people breathing for 1 minute).This can be increased to sample larger areas. After collection ofparticulates from air, the Plunging mechanism (FIG. 8-7) is activated torelease the growth media from the growth media reservoir (FIG. 1) andforce it through the filter, eluting particulate matter from the filter.In this case, we are testing for Bacillus anthracis spores. The mediashould be, appropriate for the germination of spores followed by growth,such as CA broth (Thorne and Belton, 1957. J. Gen. Microbiol.17:505-516) supplemented with 20·g/ml tryptophan and buffered with 100mM HEPES pH 8.0 (Dai and Koehler, 1997, Infection and Immunity65:2576-82).

The cartridge is then moved by the cartridge feed mechanism (FIG. 8-3)to the incubation station (FIG. 8-8). The program for the machine shouldbe set to heat shock the collected spores for 20 minutes at 65° C.,followed by a reduction in temperature to 37° C. to allow for growth ofbacteria. Incubation at 37° C. should continue for two hours or for alength of time such that the number bacteria produce enough of theelement assayed to be above the detection limit of the detection strip.

At the end of the bacterial growth stage, the device of FIG. 8 willbreak the breakable seal between the chambers of the cartridge (FIG. 4).The bacterial suspension will then flow into the chamber containing thedetection strip specific for an element of Bacillus anthracis. Once incontact with the detection strip, the suspension will be wicked up thelength of the strip past the indicator and control lines. This processshould take approximately 3-5 minutes. The results will be analyzed bythe result sensor (attached to FIG. 8-10), which will compare theindicator line to the positive control. If the indicator line and thecontrol line are positive, the test will indicate the presence ofBacillus anthracis spores in the collected sample. If the indicator lineis negative and the positive control line, is positive, then no Bacillusanthracis spores were collected in the sample. If the positive controlline is negative for any reason, the test is designated as a failure andshould be repeated. After the results have been collected, the cartridgeis moved to the Biological Waste cassette (FIG. 8-13) to store thecartridge for either re-testing or disposal.

Results from this test will be displayed as appropriate for thesituation. This could be through digital or printed results displayed atthe device, or through an integrated communication system such as a cellphone to a remote computer. In another aspect, a coupled GPS receivercould identify the location of the unit upon initiation of the sampletesting, and communicate this information as well.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

All publications, patents and patent applications cited herein arehereby incorporated by reference in their entireties for all purposes tothe same extent as if each individual publication, patent or patentapplication were specifically and individually indicated to be soincorporated by reference.

1. A sealable cartridge for detecting elements of microorganisms,comprising: (i) a sample chamber, wherein a test sample is suspended influid; (ii) a test chamber, wherein the fluid is tested for elements ofa microorganism; and (iii) a breakable seal between the sample and testchambers.
 2. The sealable cartridge of claim 1, further comprising adetection means.
 3. The sealable cartridge of claim 1, furthercomprising a transmission means.
 4. The sealable cartridge of claim 1,further comprising an assay analyzer.
 5. The sealable cartridge of claim1, wherein the microorganism is Bacillus anthracis.
 6. A microbialmonitoring system for detecting microorganisms, comprising: an intake orreceptacle through which a fluid or gas enters said microbial monitoringsystem or is added to said microbial monitoring system; a means fordrawing or pushing said fluid or gas into said microbial monitoringsystem and through conduit connectively communicating with said intake;a filtering means within said conduit through which said fluid or gaspasses and retains microorganisms present in said fluid or gas; a mediumreservoir for a growth medium on a side of said filtering means oppositefrom said intake or receptacle, wherein said growth medium passesthrough said filtering means and removes said microorganisms from saidfiltering means.
 7. The microbial monitoring system of claim 6, furthercomprising a detection means.
 8. The microbial monitoring system ofclaim 6, further comprising a transmission means.
 9. The microbialmonitoring system of claim 6, further comprising an assay analyzer. 10.A microbial monitoring system for detecting microorganisms, comprising:an intake or receptacle through which a fluid or gas enters saidmicrobial monitoring system or is added to said microbial monitoringsystem; a means for drawing or pushing said fluid or gas into saidmicrobial monitoring system, and through a conduit connectivelycommunicating with said intake; a filtering means within said conduitthrough which said fluid or gas passes and retains microorganismspresent in said fluid or gas; an exhaust chamber, wherein said filteringmeans is interposed between said intake and said exhaust chamber; amedium reservoir on a side of said filtering means opposite saidretained microorganisms, comprising a microbial growth medium and areservoir breakable seal; a plunger means, wherein pressure placed onsaid plunger means: (i) engages a hermetic seal between said mediumreservoir said filtering means; (ii) breaks said reservoir breakableseal and forces growth medium through said filtering means in adirection opposite of flow of said fluid or gas; and (iii) backwashessaid microorganisms from said filtering means and suspends saidmicroorganisms in said growth medium; an incubation chamber forincubating and growing said microorganisms; a programmabletemperature-regulating means for controlling the temperature of saidincubation chamber; an incubation chamber breakable seal through whichsaid growth medium passes into a test chamber for detecting saidmicroorganisms; a timer for controlling intervals between assaymeasurements; a detection means within said test chamber; wherein saiddetection means determines the presence or absence of saidmicroorganisms without killing said microorganisms or inhibiting saidgrowing; and a transmission means; wherein said presence or absence ofsaid microorganisms is reported via said transmission means to an assayanalyzer.
 11. The microbial monitoring system according to claims 6 or10, wherein said transmission means is a wire connection, a radio link,a microwave transmission, an infrared transmission, a cellular phone, ora computer network.
 12. The microbial monitoring system according toclaims 6 or 10, wherein said microbial monitoring system comprises anetwork of separate monitoring devices.
 13. The microbial monitoringsystem according to claim 10, wherein said assay analyzer is remote fromsaid microbial monitoring system and said reporting relays location of amicrobial monitoring device to said assay analyzer.
 14. The microbialmonitoring system according to claim 13, wherein said location isdetermined by a global positioning system.
 15. The microbial monitoringsystem according to claims 6 or 10, wherein said detection means isselected from the group consisting of an enzyme immunoassay, an opticalimmunoassay, mass spectroscopy, and gene sequence analysis.
 16. Themicrobial monitoring system according to claim 15, wherein said enzymeimmunoassay comprises an immunoassay strip that wicks said growth mediumpast a detection area and a control area on said detection strip. 17.The method according to claims 6 or 10, wherein said microorganisms areselected from the group consisting of Bacillus anthracis, Yersiniapestis, Neiserria menigitidis, Coxiella burnettii, Coccidiodes immitis,Francisella tularensis, Cryptococcosis neofomans, Escherichia coliHaemophilus influenzae, Brucella species, Salmonella species, Shigellaspecies, Chlamydia psittaci, Vibrio cholerae, Staphylococcus enterotoxinB, Histoplasma capsulation, Rickettsia species, Corynebacteriumdiphtheriae, Burkholderia pseudomallei, Burkholderia mallei,Mycobacterium tuberculosis, Blastomyces dermatitidis, Nocarida andsmallpox species.
 18. The microbial monitoring system according toclaims 6 or 10, wherein said microbial monitoring system comprises anoptical sensor for determining assay results.
 19. The microbialmonitoring system according to claims 6 or 10, wherein said microbialmonitoring system comprises a variable timer that determines thefrequency of detection assays.
 20. The microbial monitoring systemaccording to claims 6 or 10, wherein said microbial monitoring systemcomprises a detection unit comprising a disposable cartridge.
 21. Themicrobial monitoring system according to claim 20, wherein saiddisposable cartridge is moved in automated fashion into place forincubation and assay determination, said detecting is conductedautomatically, and said disposable cartridge is automatically removed toa waste chamber after said detecting.
 22. The microbial monitoringsystem according to claim 6, wherein said plunger means comprises abladed or pointed edge for releasing said growth medium.
 23. Themicrobial monitoring system according to claim 6, wherein saidprogrammable temperature-regulating means elevates said temperature ofsaid microbial monitoring system to shock bacterial endospores andinitiate endospore germination.
 24. A microbial monitoring system fordetecting microorganisms, comprising: a network of microbial monitoringdevices reporting to a remote assay analyzer, wherein each of saidmicrobial monitoring devices comprises at least one disposablecartridges comprising: an intake or receptacle through which a fluid orgas enters said microbial monitoring system or a swab is depositedwithin said microbial monitoring system in a fluid; a means for drawingor pushing said fluid or gas into said microbial monitoring system andthrough a conduit connectively communicating with said intake orreceptacle; a semipermeable membrane within said conduit through whichsaid fluid or gas passes and which retains microorganisms present insaid fluid or gas; an exhaust chamber, wherein said semipermeablemembrane is interposed between said intake and said exhaust chamber; amedium reservoir containing microbial growth medium on the side of saidsemipermeable membrane opposite said retained microorganisms; a plungermeans comprising a bladed or pointed edge for releasing said growthmedium; wherein depressing said plunger means: (i) engages a hermeticseal between said medium reservoir said semipermeable membrane; (ii)forces growth medium through said semipermeable membrane in a directionopposite of flow of said fluid or gas; and (iii) backwashes saidmicroorganisms from said semipermeable membrane and suspends saidmicroorganisms in said growth medium; a breakable seal through whichsaid growth medium passes into a test chamber for incubating and growingsaid microorganisms; a timer for determining intervals betweenimmunoassay measurements; a temperature-regulating means that maintainssaid test chamber at a temperature that promotes growth of at least onetarget microorganism; an immunoassay test strip within said testchamber; wherein said immunoassay strip wicks said growth medium past adetection area on said detection strip, said detection area comprises anindicator assay region and a control assay region; and said immunoassaytest strip determines the presence or absence of said at least onetarget microorganism; wherein said presence or absence of said at leastone target microorganism is reported with location of said microbialmonitoring device to said remote assay analyzer.
 25. A method fordetecting microbial pathogens in an air or fluid specimen, comprising:passing an air or gas specimen or a specimen derived from a swab throughthe microbial monitoring system according to claim 6; retainingmicroorganisms within said air or fluid specimen on a filtering meanswithin said microbial monitoring system; incubating said microorganismswith a growth medium in said microbial monitoring system at temperaturethat promotes growth of at least one target microorganism; anddetermining presence or absence of said at least one targetmicroorganism with a detection means within said microbial monitoringsystem; where determination of the absence of said at least one targetmicroorganism initiates a subsequent round of incubation anddetermining.
 26. A kit for detecting microbial pathogens, comprising: atleast one disposable cartridge for use in the microbial monitoringsystem according to claim 6; and a sterile container for retaining saidat least one disposable cartridge.